P-glycoprotein (Pgp) is a transmembrane pump encoded by the multidrug resistance gene, mdr1. While it is well established that Pgp in tumor cells can bind to and efflux several important chemotherapeutic drugs the physiological role(s) and substrate(s) of Pgp in normal cells are unknown. The long range goal of this proposal is to identify a functional role for Pgp in normal human lymphoid cells. This proposal is based on our recent studies demonstrating that human natural killer (NK) cells: 1) bind monoclonal antibodies specific for Pgp; 2) efflux the fluorescent compounds rhodamine 123 and DIOC2, a phenomenon associated with multidrug resistance (MDR) and mdr1 gene expression in tumor cells and 3) display significantly reduced cytotoxicity against K562 target cells and impaired ability to efflux rhodamine 123 and DIOC2 after incubation with resistance modification agents (RMA), i.e., drugs which reverse MDR activity in tumor cells. Collectively, these data suggest that Pgp is expressed in NK cells and may have a fundamental role in NK cell mediated cytotoxicity. To address these issues, we propose to inhibit Pgp function and assess the effect(s) on MDR-like activity and cytotoxicity of NK cells. Specifically, we will treat NK cells with additional RMAs, Pgp specific monoclonal antibodies or antisense oligonucleotides to Pgp mRNA, each of which can reverse MDR activity in tumor cells. MDR-like activity of NK cells will be assessed by flow cytometry, analyzing cells for the accumulation/efflux of rhodamine 123 and DIOC2 in the presence or absence of Pgp inhibitors. The effect of Pgp inhibitors on NK cell mediated cytotoxicity will be assessed by 51Cr release from labelled K562 target cells. Under conditions where cytotoxicity is inhibited, ultrastructural analysis of NK cells and their targets will be performed to determine which stage(s) in the lytic process is affected. Specifically, we will quantitatively assess the points of contact between effector and target cells as well as the distribution, orientation and exocytosis of cytotoxic granules. In addition, degranulation will be assessed by monitoring for the release of serine esterase contained within cytotoxic granules. These studies should establish a physiological role for Pgp in human NK cells and provide insight into the function of Pgp in other cell types. If inhibition of Pgp results in impaired NK cell mediated cytotoxicity, our results may have important implications regarding the ongoing clinical trials to evaluate chemosensitizing drugs as a therapy for cancer patients with multidrug resistant tumors.